Azole heteroresistance in Cryptococcus neoformans: emergence of resistant clones with chromosomal disomy in the mouse brain during fluconazole treatment.
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ABSTRACT:
SUBMITTER: Sionov E
PROVIDER: S-EPMC3811407 | biostudies-other | 2013 Oct
REPOSITORIES: biostudies-other
Publications
Azole heteroresistance in Cryptococcus neoformans: emergence of resistant clones with chromosomal disomy in the mouse brain during fluconazole treatment.
Sionov Edward E Chang Yun C YC Kwon-Chung Kyung J KJ
Antimicrobial agents and chemotherapy 20130708 10
We have previously reported that Cryptococcus neoformans strains are innately heteroresistant to fluconazole in vitro, producing minor, highly resistant subpopulations due to adaptive formation of disomic chromosomes. Using a mouse model, we assessed the emergence of heteroresistant clones in the brain during fluconazole treatment and found that the occurrence of heteroresistant clones in vivo with chromosomal disomy is strain dependent. Interestingly, emergence of heteroresistant clones in vivo ...[more]
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Project description:Heteroresistance in Cryptococcus neoformans is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, ERG11, the fluconazole target, and AFR1, a drug transporter, were reported as major factors in the emergence of Chr1 disomy. In the present study, we show Chr4 to be the second most frequently formed disomy at high concentrations of fluconazole (FLC) and characterize the importance of resident genes contributing to disomy formation. We deleted nine Chr4 genes presumed to have functions in ergosterol biosynthesis, membrane composition/integrity or drug transportation that could influence Chr4 disomy under FLC stress. Of these nine, disruption of three genes homologous to Sey1 (a GTPase), Glo3 and Gcs2 (the ADP-ribosylation factor GTPase activating proteins) significantly reduced the frequency of Chr4 disomy in heteroresistant clones. Furthermore, FLC resistant clones derived from sey1?glo3? did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to ERG11 locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in Saccharomyces cerevisiae, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in sey1? but without any discernable alteration in gcs2? and glo3? under fluorescence microscopy. The aberrant ER morphology of all three mutant strains, however, was discernable by transmission electron microscopy. A 3D reconstruction using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) revealed considerably reduced reticulation in the ER of glo3? and gcs2? strains. In sey1?, ER reticulation was barely detectable and cisternae were expanded extensively compared to the wild type strains. These data suggest that the genes required for maintenance of ER integrity are important for the formation of disomic chromosomes in C. neoformans under azole stress.
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Project description:BackgroundFluconazole is used in combination with amphotericin B for induction treatment of cryptococcal meningitis and as monotherapy for consolidation and maintenance treatment. More than 90% of isolates from first episodes of cryptococcal disease had a fluconazole minimum inhibitory concentration (MIC) ?4 ?g/ml in a Gauteng population-based surveillance study of Cryptococcus neoformans in 2007-2008. We assessed whether fluconazole resistance had emerged in clinical cryptococcal isolates over a decade.Methodology and principal findingsWe prospectively collected C. neoformans isolates from 1 January through 31 March 2017 from persons with a first episode of culture-confirmed cryptococcal disease at 37 South African hospitals. Isolates were phenotypically confirmed to C. neoformans species-complex level. We determined fluconazole MICs (range: 0.125 ?g/ml to 64 ?g/ml) of 229 C. neoformans isolates using custom-made broth microdilution panels prepared, inoculated and read according to Clinical and Laboratory Standards Institute M27-A3 and M60 recommendations. These MIC values were compared to MICs of 249 isolates from earlier surveillance (2007-2008). Clinical data were collected from patients during both surveillance periods. There were more males (61% vs 39%) and more participants on combination induction antifungal treatment (92% vs 32%) in 2017 compared to 2007-2008. The fluconazole MIC50, MIC90 and geometric mean MIC was 4 ?g/ml, 8 ?g/ml and 4.11 ?g/ml in 2017 (n = 229) compared to 1 ?g/ml, 2 ?g/ml and 2.08 ?g/ml in 2007-2008 (n = 249) respectively. Voriconazole, itraconazole and posaconazole Etests were performed on 16 of 229 (7%) C. neoformans isolates with a fluconazole MIC value of ?16 ?g/ml; only one had MIC values of >32 ?g/ml for these three antifungal agents.Conclusions and significanceFluconazole MIC50 and MIC90 values were two-fold higher in 2017 compared to 2007-2008. Although there are no breakpoints, higher fluconazole doses may be required to maintain efficacy of standard treatment regimens for cryptococcal meningitis.
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Project description:In Cryptococcus neoformans, H99 is the wild type. TJ1843 is a strains with segmental disomy of chromosome 11, which is derived from H99. In this study, approximately 1 million cells of TJ1843 were spread on YPD plate supplemented with 32 ug/ml fluconazole. Randomly 27 adaptors (TJ3305 and TJ3331) were chosen. These adaptors were sequenced.
2023-08-17 | E-MTAB-12540 | biostudies-arrayexpress